Responses to the synthetic substrate [Pro¹¹,D-Ala¹²]angiotensin I were investigated in the hindlimb vascular bed of the cat, a system in which local angiotensin-converting enzyme activity is high. Under constant-flow conditions, injections of [Pro¹¹,D-Ala¹²]angiotensin I into the perfusion circuit in doses of 1-300 µg caused dose-related increases in perfusion pressure that were rapid in onset and that were not changed by the presence of a time-delay coil in the perfusion circuit upstream from the site of peptide injection. The synthetic substrate was ~100-fold less potent than angiotensin I and II, and responses to [Pro¹¹,D-Ala¹²]angiotensin I were not altered by captopril in a dose that inhibited pressor responses to angiotensin I but did not alter responses to angiotensin II. Responses to [Pro¹¹,D-Ala¹²]angiotensin I, angiotensin I, and angiotensin II were inhibited by DUP-532 and candesartan but were not altered by the angiotensin AT₂ receptor antagonist PD-123319. The present data show that [Pro¹¹,D-Ala¹²]angiotensin I has significant vasoconstrictor activity in the hindlimb vascular bed of the cat and suggest that responses are mediated by the activation of AT₁ receptors and that activation of AT₂ receptors is not involved. The present data show that the onset of responses to [Pro¹¹,D-Ala¹²]angiotensin I and angiotensin II are similar and are not dependent on the action of the angiotensin-converting enzyme. The present data suggest that conversion of the synthetic substrate to an active peptide occurs rapidly within the hindlimb vascular bed or that the peptide may have direct AT₁ receptor-stimulating activity.